Type III Effector Activation via Nucleotide Binding, Phosphorylation, and Host Target Interaction

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Abstract
The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]–interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 Å resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function. Many bacterial pathogens use a specialized protein “injection needle” called a type III secretion system to help colonize cells of higher organisms. The type III secretion needle attaches to a host cell and is the delivery conduit for a variety of bacterial proteins that act inside of the host cell. These proteins are called type III effectors. They manipulate host cell biology in order to help the bacterial pathogen colonize the host. We studied one type III effector from plant pathogenic bacteria called Pseudomonas syringae. This effector, termed avirulence protein B (AvrB), is targeted to the inner face of the plant cell plasma membrane, where it interacts with a membrane-bound host protein called RIN4 (resistance to Pseudomonas maculicula protein–interacting protein). RIN4 is phosphorylated in the presence of AvrB and an as-yet-unknown additional host factor. We provide a structural basis for the binding of AvrB to RIN4 and a possible mechanism of action for AvrB inside the host. AvrB activation and its ability to bind RIN4 have evolved to help the pathogen, yet in Arabidopsis, the AvrB-dependent phosphorylation of RIN4 is sensed by the plant immune system, leading to a rapid halt in pathogen growth.